PROJECT SUMMARY We propose to build and analyze the first animal models of dominant spondylocarpotarsal synostosis (SCT), a human genetic disease that arises from mutations in the MYH3 embryonic myosin heavy chain. These mutations yield misshaped and fused vertebral bodies as well as carpal and tarsal abnormalities that are hypothesized to arise from primary defects in muscle function. Our transgenic Drosophila melanogaster models will be used to dissect the biochemical, biophysical, developmental and physiological bases of this disease. The Drosophila system will allow us to mutate the Drosophila myosin gene to examine the effects of two SCT alleles in a standardized genetic background. This will define the importance of interactions between wild-type and mutant myosin molecules to disease pathology and will obviate genetic heterogeneity that leads to phenotypic variability in the human disease. Further we will explore the use of our transgenic system to produce adequate quantities of human wild-type and SCT MYH3 to determine their functional properties and solve their high-resolution crystal structures. We will test the following hypotheses: that actin binding, which influences nucleotide affinity and filament motility, is abnormal in the SCT mutant myosin models; that functionally abnormal SCT myosin leads to myofibril disruption and muscle dysfunction in the Drosophila model; that structure-function relationships about human myosin can be discerned using our Drosophila-based myosin expression system. To evaluate these hypotheses, we will pursue the following specific aims: 1) Assess the ATPase, actin binding and actin motility capabilities of mutant SCT myosins compared to the wild- type protein. 2) Determine the dominant effects of the mutations on myofibrillar ultrastructure and function of muscles from the larval body wall and adult thorax (indirect flight and jump muscles). 3) Explore the possibility of producing, isolating and determining the structural and functional properties of normal and SCT human MYH3 protein using a unique indirect flight muscle expression system. This multifaceted approach will provide mechanistic insights into the molecular and developmental bases of SCT and begin to elucidate how mutations in a skeletal muscle protein lead to developmental defects in associated skeletal elements. Understanding the underlying muscle defects causative of the disease may ultimately yield therapeutic approaches.